Human immunoglobulin G or IgG antibodies exist in four subclasses, each with distinct structural and functional properties. IgGs are composed of two heavy chain-light chain pairs (half-antibodies) which are connected via inter-heavy chain disulfide bonds directly linking Cys residues in the hinge region (EU-index numbering: cysteine residues 226 and 229; Kabat numbering: cysteine residues 239 and 242). Human IgG4 molecules exist in various molecular forms which differ by the absence or presence of inter-heavy chain disulfide bonds.
A wide variety of recombinant antibody formats have been developed, such as, tetravalent bispecific antibodies by fusion of an IgG antibody format and single chain domains (Coloman et al., Nature Biotech 15 (1997) 159-163; WO 2001/077342; and Morrison, Nature Biotech 25 (2007) 1233-1234). Another format has the antibody core structure (IgA, IgD, IgE, IgG or IgM) no longer retained, such as dia-, tria- or tetrabodies, minibodies, several single chain formats (scFv, Bis-scFv). But such formats are capable of binding two or more antigens (Holliger et al., Nature Biotech 23 (2005) 1126-1136; Fischer and Leger, Pathobiology 74 (2007) 3-14; Shen et al., J. Immunological Methods 318 (2007) 65-74; and Wu et al., Nature Biotech. 25 (2007) 1290-1297).
A method for separating or preferentially synthesizing dimers which are linked via at least one inter-chain disulfide linkage from dimers which are not linked via at least one interchain disulfide linkage from a mixture comprising the two types of polypeptide dimers is reported in US 2005/0163782.
Bispecific antibodies have difficulty producing materials in sufficient quantity and quality using traditional hybrid hybridoma and chemical conjugation methods. Further, WO2005/062916 and U.S. patent application 2010/0105874 describe how to form bispecific antibodies by reducing antibody “AA” and antibody “BB” to separate the disulfide bonds into single heavy-light chain units (A or B) with a single binding region (wherein both A and B bind to different targets). Then the antibodies allow the disulfide bonds to undergo isomerization such that antibodies AB, BA, AA and BB are reformed at a probability of about 25% each. However, both AB and BA are the same bispecific antibodies and therefore represent, at best, about a 50% yield. Therefore, this requires additional steps to separate the desired bispecific antibodies formed from the original reconstituted antibodies. However, U.S. patent application 2010/0105874 points to the hinge region in IgG4 having a sequence of CPSC and stating: “the CPSC sequence results in a more flexible core hinge and the possibility to form intra-chain disulfide bonds . . . it is believed that antibodies having an IgG4-like core hinge sequence may have an intrinsic activity for rearrangement of disulfide bonds, which is simulated by the conditions used in the methods of the invention.” (paragraph 0013). In addition, other forms of bispecific antibodies have been made with a “knob and hole” structure made by altering the sequence of the heavy chains of antibodies A and B.
Therefore, the present disclosure provides a process to produce chemically-locked bispecific IgG antibodies that address the need in the art for a much higher yield of bispecific antibodies and with better stability than the knob and hole methods that alter amino acid sequences in the fixed antibody regions.